In the bacterial portion of the project, we have made progress towards structural and mechanistic elucidations of the T-box riboswitch system, as well as targeting the T-box RNA with small molecule inhibitors. Using methods that we developed in the lab to assemble functionally relevant T-box-tRNA subcomplexes, we define a T-box 3 domain that is both necessary and sufficient for specific binding with uncharged tRNAs. Using Small-angle X-ray Scattering (SAXS), we have determined a low-resolution molecular envelope of the T-box 3 domain in complex with a cognate, uncharged tRNA. These progress will facilitate high-resolution studies of the subcomplex using X-ray crystallography methods, which are currently under way. In parallel, we have been working to explore the technical feasibility to image medium-sized, protein-free RNA complexes such as a full-length T-box-tRNA complex (85 kD, no symmetry) using single-particle cryo-EM, in collaboration with Wah Chius group. In the past year, we have made significant improvements in terms of achievable resolution, and overcome preferred orientation issues encountered with certain species of T-box complexes. Lastly, working closely with the Constantinos Stathopoulos lab (University of Patras, Greece), we contributed to the characterization of interactions between mainstream antibiotics with a glycine-responsive T-box in Staphylococcus aureus in silico and in vitro. Structural modeling rationalizes the nuclease-probing data to suggest that several antibiotics bind strategic locations on the T-box stem I-tRNA structure, and thus can directly modulate the genetic outcome of the T-box regulon. This collaborative work is in press at Nucleic Acids Research. In the eukaryotic portion of the project, we are making progress in structural and mechanistic elucidations of the Gcn2 system. Gcn2 senses and manages amino acid/serum starvation, UV irradiation, oxidative/osmotic/ER stress, etc, is essential for cellular survival under stress, and is a key player in several cancers and neurodegenerative diseases. Similar to T-boxes, this multi-domain protein directly engages tRNAs and evaluates their aminoacylation status to detect nutrient limitation, and couples this readout with the activation of its dormant kinase activity. To achieve these functions, Gcn2 incorporated a domain borrowed from the Histidyl-tRNA synthetase (HisRS), the enzyme responsible for charging histidine onto tRNA-his. This represents an interesting case of enzyme repurposing and adaptation. Using an expression and purification system we have developed, we were able to isolate HisRS-like and C-terminal domains of Gcn2 in biophysical quantities. Using SAXS, we have determined a low-resolution molecular envelope of the Gcn2 HisRS-CTD dimer. This reveals that the HisRS-like domain of Gcn2 structurally mimics authentic HisRS enzyme, the parent protein from which Gcn2 has evolved. High-resolution X-ray studies of Gcn2 domains are ongoing, to understand how the HisRS enzyme evolves into the HisRS-like sensory domain of Gcn2, whilst retaining a similar overall architecture.